PT-1 selectively activates AMPK-{gamma}1 complexes in mouse skeletal muscle, but activates all three {gamma} subunit complexes in cultured human cells by inhibiting the respiratory chain
We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 on Ser555, but not proposed AMPK-γ3 substrates such as Ser231 on TBC1D1 or Ser212 on ACC2, nor did it stimulate glucose transport. Surprisingly, however, in HEK-293 cells expressing human γ1, γ2 or γ3, PT-1 activated all three complexes equally. We were unable to reproduce previous findings suggesting that PT-1 activates AMPK by direct binding between the ...
Source: BJ Signal - February 19, 2015 Category: Biochemistry Authors: T Elbenhardt Jensen, F A Ross, M Kleinert, L Sylow, J R. Knudsen, G J. Gowans, D Grahame Hardie, E A. Richter Tags: BJ Signal Source Type: research
Regulated stability of eukaryotic elongation factor 2 kinase requires intrinsic but not ongoing activity
Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical protein kinase which negatively regulates protein synthesis, is activated under stress conditions and plays a role in cytoprotection, e.g., in cancer cells. It is regarded as a possible target for therapeutic intervention in solid tumours. Earlier studies showed that eEF2K is degraded by a proteasome-depended pathway in response to genotoxic stress and that this requires a phosphodegron that includes an autophosphorylation site. Thus, application of eEF2K inhibitors would stabilise eEF2K, partially negating the effects of inhibiting its activity. Here we show tha...
Source: BJ Signal - February 11, 2015 Category: Biochemistry Authors: X Wang, J Xie, S Regufe Da Mota, C E Moore, C Gregory Proud Tags: BJ Signal Source Type: research
Functional crosstalk between Cdc42 and two downstream targets Par6B and PAK4
The establishment of polarity is an essential step in epithelial morphogenesis. Polarity proteins promote an apical/basal axis, which together with the assembly of apical adherens and tight junctions, directed vesicle transport, and the reorganization of the actomyosin filament network generate a stable epithelium. The regulation of these cellular activities is complex, but the Rho family GTPase Cdc42 is known to play a key role in the establishment of polarity from yeast to man. We previously identified two Cdc42 target proteins, the kinase PAK4 and the scaffold Par6B, required to promote the assembly of apical junctions ...
Source: BJ Signal - February 9, 2015 Category: Biochemistry Authors: D Jin, J Durgan, A Hall Tags: BJ Signal Source Type: research
An interplay between the p38 MAPK pathway and AUBPs regulate c-fos mRNA stability during mitogenic stimulation
MAPK pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes of the AP-1 family, as fos, achieve peak expression levels shortly after cells are stimulated with growth factors, and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR and KSRP, bind to a fos ARE element present in the 3’UTR region of fos mRNA regulating its stability by a still poorly defined mechanism. We show here that while HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3’UTR, KSRP counteracts this effect. Furth...
Source: BJ Signal - January 14, 2015 Category: Biochemistry Authors: M Sol Degese, T Tanos, J Naipauer, T Gringerich, D Chiappe, P Echeverria, J LaMarre, J Silvio Gutkind, O Adrian Coso Tags: BJ Signal Source Type: research
The Tribbles 2 (TRB2) pseudokinase binds to ATP and autophosphorylates in a metal-independent manner
The human Tribbles (TRB)-related pseudokinases are CAMK-related family members that have evolved a series of highly unusual motifs in the ‘pseudocatalytic’ domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl transfer to substrates. However, in pseudokinases atypical residues give rise to diverse, and often unstudied, biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks, and overexpression confers cancer phenotypes on human cells, marking ...
Source: BJ Signal - January 12, 2015 Category: Biochemistry Authors: F P Bailey, D P Byrne, K Oruganty, C E Eyers, C Novotny, K M Shokat, N Kannan, P A Eyers Tags: BJ Signal Source Type: research
TRPM2 mediated intracellular Zn2{+} release triggers pancreatic beta cell death
Reactive oxygen species (ROS) can cause pancreatic β-cell death by activating the pro-apoptotic transient receptor potential (melastatin)2 (TRPM2) channels. Cell death was attributed to the ability of these channels to raise cytosolic [Ca2+]. Recent studies, however, revealed that TRPM2 channels can also conduct Zn2+, but the physiological relevance of this property is enigmatic. Given Zn2+ is cytotoxic, we asked if TRPM2 channels can permeate sufficient Zn2+ to affect cell viability. To address this, we used the INS1 β-cell line, HEK-293 cells transfected with TRPM2 and pancreatic...
Source: BJ Signal - January 6, 2015 Category: Biochemistry Authors: P T Manna, T S Munsey, N Abuarab, F Li, A Asipu, G Howell, A Sedo, W Yang, J Naylor, D J Beech, L Jiang, A Sivaprasadarao Tags: BJ Signal Source Type: research
Conserved Proline-Directed Phosphorylation Regulates SR Protein Conformation and Splicing Function
In this study, we address the effects of discrete serine-proline phosphorylation on the conformation and cellular function of the SR protein SRSF1. Using chemical tagging and dephosphorylation experiments, we show that modification of serine-proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over one hundred human genes based on a global splicing assay. Over...
Source: BJ Signal - December 22, 2014 Category: Biochemistry Authors: M M Keshwani, B E. Aubol, L Fattet, C Ma, J Qiu, P A. Jennings, X Fu, J A. Adams Tags: BJ Signal Source Type: research
The function of phosphatidylinositol 5-phosphate 4-kinase {gamma}(PI5P4K{gamma}) explored using a specific inhibitor that targets the PI5P binding site.
NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol-5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P 4-kinase assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1μM but did not inhibit the α and β PI5P4K isoforms at concentrations up to 100μM. A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme’s ATP binding site, and direct exploration of binding using hydrogen-deuterium exchange...
Source: BJ Signal - December 11, 2014 Category: Biochemistry Authors: J H Clarke, M Giudici, J E Burke, R L Williams, D J Maloney, J J Marugan, R F Irvine Tags: BJ Signal Source Type: research
Gcn1 contacts the small ribosomal protein Rps10, which is required for full activation of the protein kinase Gcn2
In eukaryotes, amino acid deprivation leads to the accumulation of uncharged tRNAs that are detected by Gcn2, which in turn phosphorylates eIF2α, an essential process for overcoming starvation. In Saccharomyces cerevisiae, sensing amino acid shortages requires that Gcn2 binds directly to its effector protein Gcn1, and both must associate with the ribosome. Our hypothesis is that uncharged tRNAs occur in the ribosomal A-site, and that Gcn1 is directly involved in transfer of this starvation signal to Gcn2. Here we provide evidence that Gcn1 directly contacts the small ribosomal protein Rps10. Gcn1 residues 1060-1777 ...
Source: BJ Signal - December 1, 2014 Category: Biochemistry Authors: S Lee, M J Swanson, E Sattlegger Tags: BJ Signal Source Type: research
Decreased adipogenesis and adipose tissue in mice with inactivated protein phosphatase 5
Glucocorticoids play an important role in the treatment of inflammation and immune disorders, despite side effects, which include metabolic derangements such as central adiposity. These studies examine the role of protein phosphatase 5 (Ppp5) in glucocorticoid receptor complexes, which mediate response to glucocorticoids. Mice homozygous for inactivated Ppp5 (Ppp5D274A/D274A) exhibit decreased adipose tissue surrounding the gonads and kidneys compared with wild type mice. Adipocyte size is smaller, more preadipocytes/stromal cells are present in their gonadal fat tissue and differentiation of preadipocytes to adipocytes is...
Source: BJ Signal - December 1, 2014 Category: Biochemistry Authors: W Jacob, D Rosenzweig, C Vázquez-Martin, S L Duce, P T.W. Cohen Tags: BJ Metabolism Source Type: research
Raptor ablation in skeletal muscle decreases Cav1.1 expression and affects the function of the excitation-contraction coupling supramolecular complex
This study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction coupling is affected by mTORC1 signaling. (Source: BJ Signal)
Source: BJ Signal - November 28, 2014 Category: Biochemistry Authors: R J Lopez, B Mosca, S Treves, M Maj, L Bergamelli, J C Calderon, C Florian Bentzinger, K Romanino, M N. Hall, M A. Rüegg, O Delbono, C Caputo, F Zorzato Tags: BJ Signal Source Type: research
Transmembrane Oligomeric form of Vibrio cholerae Cytolysin Triggers TLR2/TLR6-dependent Pro-inflammatory Responses in Monocytes and Macrophages
Vibrio cholerae cytolysin (VCC) kills target eukaryotic cells by forming transmembrane oligomeric β-barrel pores. Once irreversibly converted into the transmembrane oligomeric form, VCC acquires unusual structural stability, and loses cytotoxic property. It is therefore possible that upon exerting its cytotoxic activity, oligomeric form of VCC retained in the disintegrated membrane fractions of the lysed cells would survive within the host cellular milieu for sufficiently prolonged duration, without causing any further cytotoxicity. Under such circumstances, VCC oligomers may potentially be recognized by the host im...
Source: BJ Signal - November 28, 2014 Category: Biochemistry Authors: B Khilwani, A Mukhopadhaya, K Chattopadhyay Tags: BJ Signal Source Type: research
Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a G{alpha}i/o protein-biased signaling cascade by Drosophila sex peptide
In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide BmPTSP, isolated from the larval brain of Bombyx mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide has been identified as a receptor for BmPTSP. However, details on the signaling pathways and physiological functions of this receptor have remained elusive. Here, we report the functional characterization...
Source: BJ Signal - November 25, 2014 Category: Biochemistry Authors: X He, J Zang, H Yang, H Huang, Y Shi, C Zhu, N Zhou Tags: BJ Signal Source Type: research
Neutral Sphingomyelinase-2 is a Redox Sensitive Enzyme:Role of Catalytic Cysteine Residues in Regulation of Enzymatic Activity through Changes in Oligomeric State
In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state. (Source: BJ Signal)
Source: BJ Signal - October 7, 2014 Category: Biochemistry Authors: P Patrick Dotson II, A A. Karakashian, M N. Nikolova-Karakashian Tags: BJ Biomolecules Source Type: research
The Crystal Structure of the RhoA : AKAP-Lbc DH-PH Domain Complex
The RhoGEF domain of AKAP-Lbc (AKAP13) catalyses nucleotide exchange on RhoA and is involved in development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA:GDP and the AKAP-Lbc RhoGEF (DH-PH) domain to 2.1 Å resolution. The structure reveals important differences compared to related RhoGEF proteins such as Leukemia-associated RhoGEF. Nucleotide exchange assays comparing the activity of the DH-PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained...
Source: BJ Signal - September 4, 2014 Category: Biochemistry Authors: K R Abdul Azeez, S Knapp, J M. P. Fernandes, E Klussmann, J M Elkins Tags: BJ Biomolecules Source Type: research
